JPH0833395B2 - Method for producing molded article for solid-phase immunoassay - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a molded article such as a container for a sample or a carrier, which is used in a solid-phase immunoassay method which is one method of immunoassay.

[Prior art]

In recent years, analytical methods using immune reactions have been adopted in a wide range of fields in medicine, and quantification of drugs in blood, detection of cancerous proteins, determination of histocompatibility, etc. have been carried out, giving extremely useful clinical data. There is. In addition, in each field of biotechnology, which is currently showing remarkable development, the analytical method using this immune reaction is also adopted. For example,
This method is indispensable, such as detection of physiologically active substances produced by gene conversion and identification of viruses that infect crops.

There are various analytical methods utilizing the immune reaction. That is, immunodiffusion, immunochromatography,
Immunoelectrophoresis, complement fixation reaction, hemagglutination reaction, passive hemagglutination reaction, fluorescent antibody method, radioimmunoassay, enzyme immunoassay and the like. Among these, a method of immobilizing an antigen or antibody on a solid phase for measurement is called a solid phase immunoassay, and for example, a solid phase radioimmunoassay or
Enzyme Linked Immunosorbent As
say, hereinafter referred to as ELISA). This solid-phase immunoassay is the most sensitive quantification method among many immunoassays. In this measuring method, an antigen or antibody is immobilized on a solid phase in advance, and an antigen-antibody reaction is performed with the antibody or antigen to which a labeling substance such as a radioisotope, a fluorescent substance or an enzyme is bound. The reaction product and unreacted product can be easily separated by washing, and the antigen or antibody immobilized on the solid phase surface by the antigen-antibody reaction is
It can be quantified by measuring the labeled substance.

In this assay, a large amount of antigen or antibody is added to the solid phase,
In addition, it is important to secure and fix with good stability.
The amount of antigens and antibodies immobilized on the surface of the solid phase carrier was small and not satisfactory.

Therefore, studies have been conducted to make it easier for the antigens and antibodies to be adsorbed and fixed by applying some treatment to the solid-phase surface. For example, treating the polystyrene polymer measurement carrier in fuming sulfuric acid or sulfuric anhydride. Sulfonic acid groups were introduced by the method to increase the amount of immobilized antigens and antibodies, and a solid phase carrier for immunoassay (JP-A-59-84161) was used to modify the surface chemistry of the substrate by plasma-treating the substrate surface. And controlling the binding and / or orientation of large molecules to increase the binding amount of antibodies and the like (JP-A-59-80442).
Gazette) is disclosed.

However, even with these treatments, the amount of the antigen or antibody adsorbed and immobilized on the solid surface cannot be said to be sufficient, and since the effect varies depending on the treatment method and conditions, the amount of adsorption and immobilization is further improved. Stabilization is required.

[Object of the Invention]

The present invention solves the drawbacks of conventional molded articles for solid-phase immunoassay, that is, that the adsorptivity of the antigen is not sufficient, and is not limited to merely examining the material and the method of chemical surface treatment. As a result of earnestly proceeding with physical and electrical studies, the present invention has been completed. The purpose is to manufacture molded articles for immunoassays that can increase the adsorption stability of antigens or antibodies (proteins, glycoproteins, peptides, etc.), which had previously been poorly adsorbed and could not be measured. It is in the process of providing a method.

[Structure of Invention]

The molded article for solid phase immunoassay according to the present invention for achieving the above object has a surface of a polystyrene molded article or a polypropylene molded article having an oxygen atom / carbon atom ratio of 0.07 to 0.19 determined from X-ray photoelectron spectrum. The structural feature is that the oxidation treatment is performed so as to be within the range.

As the plastic constituting the molded article for solid phase immunoassay of the present invention, polystyrene or polypropylene is selectively used. The shape of the molded product is naturally determined by the immunoassay method and is not particularly limited. Test tubes, beads, microplates, cuvettes, disks and the like are good examples.

The oxygen atom / carbon atom ratio on the surface of such a plastic molded article is adjusted to be in the range of 0.07 to 0.19 by chemical treatment with a chemical such as acid, alkali or peroxide, corona discharge treatment, There are low-temperature plasma treatments, irradiation with electron beams and gamma rays, etc., but they are not particularly limited.

Regarding the adsorption phenomenon of proteins on glass, plastic, etc., various studies have been conducted in the field of chromatography used for the separation and purification of proteins, but the forces that contribute to the adsorption are hydrophobic bonds, ionic bonds and hydrogen. Bonding is believed to be the main force of action. The conventional molded articles for solid-phase immunoassays mainly adsorb proteins by utilizing hydrophobic bondability or covalent bond property for chemically fixing. However, the protein to be adsorbed is a polymer having a large number of amino groups, carboxyl groups, hydroxyl groups, carbonyl groups, etc. in the molecule, and it is better to add ionic bond or hydrogen bond action force than the case where only hydrophobic bond force is applied. It is clear that the adsorptive fixing ability is increased.

The present invention is to oxidize the surface of a molded article made of polystyrene or polypropylene to try to contribute to the adsorption of not only hydrophobic bond but also ionic bond force and hydrogen bond force. Depending on the treatment conditions, the adsorption degree of protein can be fixed by adjusting the degree of oxidation so that the oxygen atom / carbon atom ratio obtained from the X-ray photoelectron spectrum of the surface of the molded article is in the range of 0.07 to 0.19. Is a significant increase. When the oxygen atom / carbon atom ratio is less than 0.07, the ionic bond force and the hydrogen bond force do not sufficiently contribute to the adsorption, and when it is 0.19 or more, that is, when the oxidation treatment is excessively performed, the oxidation degree becomes too strong. Therefore, the hydrophobic binding property of the protein is suppressed, and a special protein,
For example, the adsorptivity of basic proteins increases, but the adsorptivity generally decreases.

There are various kinds of proteins to be subjected to the immunoassay, and therefore it is desirable to prepare various types of oxidation states on the surface of the molded article for immunoassay, according to the present invention. Of the oxygen atoms /
By adjusting the ratio of carbon atoms, it is possible to prepare an immunoassay molded article having a certain protein adsorption property according to the purpose of use.

In addition, chemical treatment with chemicals such as acids and alkalis requires selection of reagents depending on the plastic material.
After the treatment, it is necessary to wash the reaction solution sufficiently. Compared with the dry method which is a physical treatment, this treatment method has an effect of roughening the surface at a microscopic level in addition to the oxidation of surface atoms, and has a characteristic of further increasing the adsorptivity. on the other hand,
Physical treatments such as corona discharge, low temperature plasma treatment, electron beam irradiation, and gamma ray irradiation can oxidize only the surface atoms and form a uniform surface state.

〔The invention's effect〕

The molded article for solid-phase immunoassay obtained by the present invention is excellent in adsorption immobilization of antigen or antibody and gives good immunological analysis results, and is extremely useful in the fields of medical diagnostics, research fields of biotechnology, etc. It is something. Further, it can be applied to any shape regardless of the shape of the molded product.

 Hereinafter, the present invention will be described with reference to examples.

Example 1 A polystyrene 96-well multiplate (manufactured by Sumitomo Bakelite Co., Ltd.) was prepared and subjected to no treatment, gamma ray irradiation of 3.0, 6.0, 8.0 and 15.0 Mrad, low temperature plasma treatment (air pressure 0.2). Torr, frequency 13.56
(5 MHz, output 50 W) for 5 and 10 minutes, and low temperature plasma treatment for 7 minutes, and then dipping treatment in 5% hydrogen peroxide solution at 40 ° C for 60 minutes. ~ Sample (8). A tissue culture plate manufactured by Flow Co. was used as a comparative sample.

For each of these samples, X made by ULVAC-PHI
The oxygen atom / carbon atom ratio on the sample surface was determined from the X-ray photoelectron spectrum measured by a line photoelectron analyzer.

On the other hand, the evaluation of the adsorptivity to the antibody (protein) was performed as follows. First, in each well of the plate, the alkaline phosphatase labeled immunoglobulin G (antibody) (Mi
100 μl / well of phosphate buffer diluted 1000 times and 10000 times respectively, and adsorbed at 37 ° C. for 60 minutes, and then Tween 20 (Tokyo Kasei) 0.05% physiological saline solution (Hereinafter, it is called a washing solution), and washed three times to remove unadsorbed components on the plate. Next, a substrate solution (0.05-M carbonate buffer solution containing 2-sodium phenylphosphate 3.8 mM, 4-aminoantipyrine 5.4 mM, and magnesium chloride 50 nM) was added at 100 µl / well, and the enzyme reaction was performed at 37 ° C for 60 minutes. It was 100 μl / 36 mM potassium ferricyanide solution
After adding a well to stop the reaction, the absorbance of the reaction solution was measured with light having a wavelength of 492 nm.

The measurement results are as shown in Table 1, and by adjusting the oxygen atom / carbon atom ratio to 0.0714 to 0.1357 and performing the oxidation treatment, the absorbance, that is, the adsorptivity to the antibody increases,
It can be seen that when the oxygen atom / carbon atom ratio is 0.318, the oxidation is insufficient and the absorbance does not increase sufficiently, while when the ratio is 0.231, the oxidation proceeds too much and the adsorptivity decreases. In addition, since the plate made by Flow Co. used for comparison has a large oxygen atom / carbon atom ratio of 0.32, it is recognized that the absorbance is extremely decreased.

Example 2 A 5 ml polypropylene tube manufactured by Sumitomo Bakelite Co., Ltd. was subjected to low temperature plasma treatment in argon gas for 20 seconds (pressure 0.2 Torr, frequency 13.56 MHz, output 50 W). The oxygen atom / carbon atom ratio on the surface was measured in the same manner as in Example 1.

Human albumin in physiological saline was 1, 10, 10 respectively.
A solution adjusted to a concentration of 0 μg / ml was added to the tube in an amount of 500 μl and allowed to adsorb at 37 ° C. for 60 minutes. Wash 3 times with washing solution and add 600 μl of 1% gelatin solution as blocking operation at 37 ℃
And reacted for 60 minutes. After washing three times with a washing solution, anti-human albumin antibody (peroxidase labeled, manufactured by Capel)
500 μl was added and reacted with the antigen, human albumin. After washing and removing the unreacted portion, the substrate solution (2,2′-azinodi (3-ethylbenzthiazoline) -6′-sulfonic acid diammonium salt 2.5 mM, hydrogen peroxide solution 5 mM) was added to 500
μl was added and reacted at 37 ° C. for 20 minutes. After stopping the reaction with 100 μl of 0.05% sodium nitride, the absorbance was measured at a wavelength of 405 nm with a flow cell type spectrophotometer.

The measurement results are as shown in Table 2, and the molded tube surface-oxidized so that the ratio of oxygen atoms / carbon atoms by plasma treatment was 0.190 showed a significant increase in absorbance compared to the untreated tube. Is recognized.

Claims (1)

[Claims] 1. A solid phase characterized by subjecting the surface of a polystyrene molded product or a polypropylene molded product to an oxidation treatment such that the oxygen atom / carbon atom ratio determined from X-ray photoelectron spectra is in the range of 0.07 to 0.19. Manufacturing method of molded article for immunoassay.